This application relates generally to methods for distinguishing and sorting cells. In particular, this application relates to methods for differentiating post-mitotic (or post meiotic) eukaryotic and prokaryotic daughter cells due to differences in the cells' DNA, RNA, chromatin associated proteins, or other distinguishable cellular features. The cells may then be sorted according to the differential cellular features.
The Applicant has invented tissue preparation compositions and methods that dramatically improve visualization of cellular and intracellular morphology and function, including evaluation of differentials in post-mitotic cell pairs; evaluation of differences in chromosomes during mitosis; improved identification of detailed histochemical, proteomic, RNA and enzymatic features of cellular organelles, such as mitochondria and the nucleus; evaluation of differential viral infections; and improved cancer diagnosis and gradation. Features of the tissue preparation compositions and methods are disclosed in U.S. patent application Ser. No. 11/400,468 filed on Apr. 17, 2006 entitled “Compositions and Methods for Preparing Specimens for Microscopic Analysis,” the entire disclosure of which is incorporated herein by reference.
The foregoing patent application includes examples and photomicrographs that illustrate differential staining of post-mitotic cell pairs. Such photomicrographs have led the Applicant to discover, contrary to the generally accepted dogma, that post-mitotic eukaryotic daughter cells vary due to differences in the cells' chromatin. In order to understand these differences, this application provides a brief explanation of chromatin.
To avoid being severely tangled or broken, DNA is packaged and wound in a complex that contains histones and nonhistone proteins. This complex, called chromatin, is generally found in the nucleus of eukaryotic cells and is the fundamental packaging unit from which chromosomes are made. Within a cell's chromatin, DNA is wound around a group of histones, known as the octomeric core, or octomer, and forms complexes known as nucleosomes, which are responsible for the “beads on a string” appearance of chromatin frequently observed in electron micrographs.
Histones are relatively small proteins with a very high proportion of positively charged amino acids (lysine and arginine) near their N-terminal end. The positive charge of these amino acids may help the highly negatively charged DNA bind tightly to histones in the octomer. The octomer generally comprises eight nucleosomal histones, or two units of each of the following histones: H2A, H2B, H3, and H4. Nearly two full turns of DNA are wound (83 nucleotide pairs per turn) around each octomer to form a nucleosome. Additionally, histone H1 binding (with multiple other factors) between nucleosomes increases the density of the DNA in chromatin. Chromatin may also contain other proteins, including the high mobility group (“HMG”) chromosomal proteins HMG 14 and HMG 17 that may be bound to nucleosomes.
Further, the proteins found in the chromatin (e.g., histone H2A, H2B, H3, and H4, HMG 14, HMG 17, and H1) (“chromatin proteins”) may also be modified. In some cases, various amino acids of the proteins may be phosphorylated, methylated, acetylated, and in some may be ribosylated or sumoylated. For example, histone H4 may be acetylated at lysine 16, lysine 8, or histone 1 may be phosphorylated. These protein modifications may also vary between daughter cells.
The Applicant has discovered that although two post-mitotic daughter cells have originated from the same cell division and have identical DNA (or nearly identical DNA as in differential DNA methylation), the two post-mitotic daughter cells are dimorphic, or asymmetrical, and can be sorted into two classes according to differences in the cells' chromatin, proteome, glycoproteome, RNA, DNA methylation, or other distinguishable cellular feature. In particular, one type of cell, which may be called class 0 for ease, seems to have chromatin that is lightly clumped in the periphery and finely divided in the center. Chromatin from these class 0 cells tends to stain lightly, as compared to the other class of cells. Additionally, the nucleolus in these cells tends to be centrally-located. The second class, which may be called class 1 for ease, has chromatin that is more densely clumped at the periphery with larger dense clumps in the inner portion and tends to stain darker than the class 0 cells. It is presumed that, in some respects, the differential staining exhibited in the two classes of cells is due to differential modification of histones.
Because histone proteins may be modified, and because the two classes of daughter cells generated from a cell division may contain differential modifications of histone proteins, the DNA in the two daughter cells may be differentially bound in chromatin. This difference in genome packaging may influence expression and activity of the packaged DNA, and as a result, influence the RNA, proteins, and glycoproteins that a cell produces, as well as the functions of a cell. For example, class 0 cells seem to express more genes than do class 1 cells. Because class 0 and class 1 cells express different numbers of genes, they ultimately express a different number, and likely type, of proteins.
Another nonbinding theory that may be used to explain dimorphic chromatin and other differences found in the two classes of cells is that epigenetic differences may be due to asymmetric methylation of the duplicated DNA that is found in daughter cells. The most common eukaryotic DNA modification is methylation of cytosine at position 5 (“m5C”). In animals, cytosine residues at CpG dinulceotides are often the preferred targets for DNA methylation, while methylation at CpG, CpN, and CpNpG sequences is common in plants. Often, methylation of the CpG, CpN, and CpNpG sites between parent and daughter DNA is not completely symmetrical. This asymmetry in DNA methylation between daughter cells may be due to many factors, including RNA-RNA interactions, and RNA-DNA interactions that serve as a signal to trigger de novo DNA methylation. For a review of this RNA-directed DNA Methylation see Theirry Pélissier et al., Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA Methylation, Nucleic Acids Research, 1999, Vol. 27, No. 7.
In both plants and animals, DNA methylation has been identified as a powerful mechanism to regulate gene expression and is thought to play an essential role in a number of cellular processes, such as developmental control, genomic imprinting, and gene silencing. Without being bound by theory, nonsymmetrical methylation of DNA between daughter cells may be partially responsible for the differences between the two classes of cells.
Additionally, the Applicant has discovered that post-mitotic daughter cells tend to be spatially entangled. For example, the cells are often arranged spatially so that each cell type is either directly adjacent to cells, or separated by only one or a few cells, from the other cell class. In this manner, cellular mediators produced by each class of cells may be readily transmitted to cells of the other class. Other means of cellular communication may also be transmitted to cells of the other class.
The Applicant has further discovered that these two classes of post-mitotic daughter cells seem to be temporally entrained, or synchronized. For instance, the Applicant has noticed that if one of the two daughter cells from a mitotic division is killed, the other cell will usually divide and form a new pair of cells or enter apoptosis. In addition, cell pairs tend to apoptose together. It is hypothesized that if live daughter cells are separated, the daughter cells divide to form a new pair of cells.
Irrespective of the theories that explain the reasons for the differences between the two classes of cells, this discovery of the two classes of cells and their different characteristics open vast fields of scientific research. Therefore, it will be appreciated that there is a need in the art for methods to distinguish and sort post-mitotic daughter cells into classes in order to enable scientific research of the binary cellular operating system.